Optically based biomolecular assay techniques such as optical microtiter plate reading and optical molecular imaging are powerful tools for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vitro and in vivo. These techniques have been increasingly used to probe protein function and gene expression. Optically based techniques exhibit advantages of picosecond temporal resolution which is important for functional imaging, submicron spatial resolution, in vivo microscopy, single molecule sensitivity and other minimally invasive techniques. These techniques also offer the potential for simultaneous use of multiple and distinguishable probes in molecular imaging. By eliminating ionizing radiation, these techniques also offer safety. These techniques have advanced over the past decade due to rapid developments in laser technology, sophisticated reconstruction algorithms and imaging software originally developed for non-optical, tomographic imaging modes such as CT and MRI.
Of the various optical imaging techniques investigated to date, near-infrared fluorescence (NIRF) imaging is of particular interest for non-invasive in vivo imaging because of the relatively low tissue absorbance, minimal autofluorescence of near-infrared (NIR) light, and deep tissue penetration of up to 6-8 centimeters. In near-infrared fluorescence imaging, a laser or appropriately filtered light is used as a source of fluorescence excitation. The excitation light travels through body tissues. When it encounters a near-infrared fluorescent molecule (“contrast agent” or “probe”), the excitation light is absorbed. The fluorescent molecule then emits light as fluorescence with a longer wavelength and therefore spectrally distinguishable from the excitation light. Despite good penetration of biological tissues by near-infrared light, conventional near-infrared fluorescence probes are subject to many of the same limitations encountered with other contrast agents, including low signal-to-noise ratios.
A number of NIRF contrast-enhanced optical imaging probes have been developed and evaluated in small animals. These studies have established the use of near-infrared optical imaging in diagnosis, molecular characterization, and monitoring of treatment response in a number of disease models. Nanoparticles have been increasingly used in a wide range of biomedical applications such as drug carriers and imaging agents. They are engineered materials with dimensions typically smaller than 100 nm, which are small enough to reach almost anywhere in the body. These nanoparticles can be derivatized with a variety of targeting ligands, multiple imaging moieties for multiple modalities imaging, or loaded with multiple molecules of a contrast agent, providing a significant boost in signal intensity for diverse imaging modalities. NIRF imaging based on nanoparticulate imaging probes is rapidly emerging as an advanced technology for noninvasive cancer detection, diagnostic and therapeutic applications. Because small probes are subjected to fast excretion in vivo, given internal clearance of small molecules and reticuloendothelial system clearance of non-immunologically shielded compounds, nanoparticle-based imaging probes offer potential advantages over small molecule or other low molecular weight polymer-based probes.
The principle of detection by fluorescence resonance energy transfer (FRET), also known as Förster resonance energy transfer (FRET), resonance energy transfer (RET), and electronic energy transfer (EET), is based on the transfer of energy from an excited donor dye to an acceptor dye or quenchers that are located in spatial proximity. Dark acceptors or quenchers are substances that absorb excitation energy from a fluorophore and dissipate the energy as heat; while fluorescent acceptors or quenchers re-emit much of this energy as light. FRET can be used to determine distance at a molecular level in a range between approximately 1 to 10 nm because the efficiency E of the energy transfer is very sensitive to the distance R between the donor species and acceptor species and declines proportionally to R06/(R06+R6), where R0 is the material-specific Förster radius defined as the distance at which the efficiency is 50%, and typically lies in the range of a few nanometers (less than approximately 10 nm). Depending on the fluorescence quantum efficiency of the acceptor species, the energy transferred from the donor species to the acceptor species can either undergo nonradiative relaxation by means of internal conversion thereby leading to quenching of the donor energy, or can be emitted by means of fluorescence of the acceptor species.
FRET occurs between the electronic excited states of the donor species and acceptor species when they are in sufficient proximity to each other, in which the excited-state energy of the donor species is transferred to the acceptor species. The result is a decrease in the lifetime and a quenching of fluorescence of the donor species. In one application of this principle, a fluorescent moiety is caused to be in close proximity to a quencher moiety. In this configuration, the energy from the excited donor fluorescent moiety is transferred to the acceptor quencher moiety and dissipated as heat rather than fluorescence.
The use of fluorescence resonance energy transfer (FRET) labels in biological systems is known. The principle has been used in the detection of binding events or cleavage reactions in assays employing fluorescence resonance energy transfer. In the case of peptide cleavage reactions, a fluorescent donor dye and fluorescent acceptor dye may be attached to a peptide substrate on either side of the peptide bond to be cleaved and at such a distance that non-radiative energy transfer between the donor and the acceptor species takes place. For example, EP 0428000 discloses a novel fluorogenic peptide substrate involving a fluorescent donor molecule and a quenching acceptor molecule attached thereto. The labeled substrate can be used in the detection and assay of a viral protease enzyme, whereby, if there is enzyme present in a test sample, the substrate is cleaved and the donor and acceptor species are thereby separated. The resultant fluorescent emission of the donor species can be measured.
In biological systems, FRET is used to detect the mutual spatial proximity of appropriately labeled biomolecules or particles. FRET can be used as a method for detecting protein-protein interactions, e.g., as a method for detecting an antigen-antibody reaction, a receptor-ligand interaction, a nucleic acid hybridization, hormone-receptor interaction or the binding of proteins to nucleic acids. The detection is itself effected by means of measuring the change in the intensity of, or the spectral change in, the donor fluorescence or acceptor fluorescence, or by means of measuring a change in the decay time of the donor fluorescence. While such systems may have achieved certain degrees of success in their particular applications, there remains a need for an activatable imaging probe with a suitable signal-to-noise ratio for use in vivo and in vitro.